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FLT3 Signalling Pathways in Acute Myeloid Leukaemia

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FLT3 Signalling Pathways in Acute Myeloid Leukaemia FLT3 signalling pathways in Acute Myeloid Leukaemia A thesis submitted to the University of Manchester for the degree of Doctor of Medicine In the Faculty of Biology, Medicine and Health 2019 Claudia M. Gorcea School of Medicine CONTENTS CONTENTS....................................................................................................................................... 2 LIST OF FIGURES .......................................................................................................................... 4 LIST OF TABLES ............................................................................................................................ 6 ABBREVIATIONS ........................................................................................................................... 7 DECLARATION............................................................................................................................... 9 COPYRIGHT STATEMENT .......................................................................................................... 9 ACKNOWLEDGEMENT .............................................................................................................. 10 ABSTRACT ..................................................................................................................................... 11 1 INTRODUCTION ................................................................................................................... 12 1.1 ACUTE MYELOID LEUKAEMIA (AML) ............................................................................... 12 1.1.1 Introduction ................................................................................................................... 12 1.1.2 Pathogenesis ................................................................................................................. 12 1.1.3 Clinical presentation and diagnosis ............................................................................. 13 1.1.4 Classification ................................................................................................................ 14 1.1.5 Prognostic factors and groups ...................................................................................... 15 1.1.6 Treatment ...................................................................................................................... 18 1.2 FLT3 MUTATIONS ............................................................................................................. 22 1.2.1 Introduction ................................................................................................................... 22 1.2.2 Types of Mutations ........................................................................................................ 23 1.2.3 Signalling pathways through FLT3 ............................................................................... 26 1.2.4 FLT3 mechanism of action ............................................................................................ 29 1.2.5 FL background .............................................................................................................. 30 1.2.6 FLT3 and impact on AML behaviour ............................................................................ 32 1.3 FLT3 TKIS OVERVIEW ...................................................................................................... 33 1.3.1 History ........................................................................................................................... 33 1.3.2 Clinical trials of FLT3 TKIs in monotherapy ............................................................... 35 1.3.3 FLT3-TKIs in combination with chemotherapy ............................................................ 39 1.3.4 FLT3-TKIs in combination with other small molecules ................................................ 39 1.3.5 FLT3 TKIs and role post stem cell transplantation ...................................................... 40 1.3.6 Molecular mechanisms of resistance to TKIs ............................................................... 40 1.3.7 Signalling pathways related to FLT3 ............................................................................ 43 1.4 MASS SPECTROMETRY ...................................................................................................... 50 1.5 HYPOTHESIS ...................................................................................................................... 58 1.6 AIMS .................................................................................................................................. 58 2 MATERIALS AND METHODS ........................................................................................... 59 2.1 MATERIALS ....................................................................................................................... 59 2.1.1 Reagents and buffers ..................................................................................................... 59 2.1.2 Cell lines ....................................................................................................................... 62 2.1.3 Drugs ............................................................................................................................. 62 2.2 METHODS .......................................................................................................................... 63 2.2.1 Cell culture techniques .................................................................................................. 63 2.2.2 Analytical techniques .................................................................................................... 65 2 3 RESULTS AND ANALYSIS.................................................................................................. 73 3.1 SIGNALLING AND CELL BIOLOGICAL EFFECT ANALYSIS ................................................. 74 3.1.1 Identification of optimal components for FLT3 signalling studies ............................... 74 3.1.2 Examination of response to FL ..................................................................................... 80 3.1.3 Selection of TKI and cell response to FLT3 inhibition or stimulation .......................... 86 3.1.4 Morphological and functional cell response to FLT3 inhibition .................................. 89 3.2 PROTEOMIC ANALYSIS .................................................................................................... 100 3.2.1 Introduction ................................................................................................................. 100 3.2.2 Proteomics analysis of biologically relevant differences in protein expression between MV411 and OCI-AML3 in relation to FLT3 stimulation or inhibition ........ 101 3.2.3 Proteomic analysis of the differences in the nature of the proteins expressed by MV4-11 and OCI-AML3 cells during Quizartinib treatment ................................ 110 3.2.4 Analysis of individual proteins linked to specific biological responses in order to identifycandidate proteins for clinical exploitation ...................................... 117 4 CONCLUSIONS AND DISCUSSION ................................................................................ 122 4.1 SIGNALLING AND CELL BIOLOGICAL EFFECT EXPERIMENTS ......................................... 123 4.2 PROTEOMIC ANALYSIS .................................................................................................... 125 5 FUTURE WORK .................................................................................................................. 128 REFERENCES .............................................................................................................................. 129 APPENDIX 1 - OVERVIEW OF CURRENTLY AVAILABLE ACUTE MYELOID LEUKAEMIA (AML) HUMAN CELL LINES AND THEIR CHARACTERISTICS. ............................................................................................ 146 APPENDIX 2 - LIST OF THE TOTALITY OF PROTEINS IDENTIFIED FROM THE GENERATED PROTEOMIC DATA, UTILISING THE STRING TOOL. ...... 147 APPENDIX 3 - CHANGES IN PROTEIN EXPRESSION IN OCI-AML3 AND MV4-11 CELL LINES. SWATH-MS ANALYSIS RAW DATASETS ............... 152 APPENDIX 4 - LIST OF PROTEIN IDENTITIES FROM APOPTOSIS PATHWAY DOWNREGULATED BY THE COMBINATION OF QUIZARTINIB AND FL IN MV4-11 CELL LINE .................................................................................... 153 APPENDIX 5 - LIST OF PROTEIN IDENTITIES FROM THE CELL CYCLE PATHWAY UPREGULATED BY THE COMBINATION OF QUIZARTINIB AND FL IN MV4-11 CELL LINE .............................................. 156 Total word count: 32259 3 LIST OF FIGURES Figure 1. The molecular and cytogenetic landscape in AML. .......................................................... 17 Figure 2. Schematic representation of the FLT3 receptor with bound FLT3 ligand (FL). ............... 24 Figure 3. Protein phosphorylation and signal cascade downstream from activated FLT3. .............. 27 Figure 4. Schematic summary of the FLT3 signalling pathway and altered gene regulation ........... 28 Figure 5. Representation of the juxtamembrane domain configuration of FLT3. ............................ 30 Figure 6. Representation of a FLT3 molecule and the potential mechanism of inhibition through TKI. ................................................................................................................... 37 Figure 7. Schematic representation of the different types and mechanisms
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